Fecal microbiota and metabolomics as preclinical diagnostic biomarkers in neonatal microbiome-driven diseases

This thesis focusses on the potential of gut microbiota and fecal volatile organic compounds (VOC) as novel preclinical biomarkers for microbiome-driven neonatal diseases. Fecal volatile organic compounds are hypothesized to reflect the function of the gut microbiota, host and interaction between both. Previous studies have demonstrated that preterm infants who developed late onset sepsis (LOS), can be differentiated from non-LOS cases based on their fecal VOC profile. In this thesis, it has been demonstrated that LOS cases can be differentiated from controls based on their gut microbiota composition in a preclinical phase. Necrotizing enterocolitis (NEC), a devastating gastro-intestinal disease, occurring in preterm infants, could also be differentiated from controls based on their fecal VOC profiles and microbiota composition. This difference was mostly profound in severe NEC. Before clinical implementation of fecal VOC as preclinical biomarker, a uniform analytical protocol should be developed. Therefore, potential influencing factors on fecal VOC outcome were assessed. This thesis proposes an analytical protocol for fecal VOC analysis in preterm born infants

Optimized sampling conditions for fecal volatile organic compounds analysis by means of field asymmetric ion mobility spectrometry

Background Fecal volatile organic compounds (VOCs) are increasingly considered as potential non-invasive, diagnostic biomarkers for various gastrointestinal diseases. Knowledge of influence of sampling conditions on VOC outcomes is limited. We aimed to evaluate effects of sampling conditions on fecal VOC profiles and to assess under which conditions an optimal diagnostic accuracy in the discrimination between pediatric inflammatory bowel disease (IBD) and controls could be obtained. Methods Fecal samples from de novo treatment-naïve pediatric IBD patients and healthy controls (HC) were used to assess effects of sampling conditions compared to the standard operating procedure (reference standard), defined as 500mg of sample mass, diluted with 10mL tap water, using field asymmetric ion mobility spectrometry (FAIMS). Results A total of 17 IBD (15CD and 2 UC) and 25 HC were included. IBD and HC could be discriminated with high accuracy (accuracy=0.93, AUC=0.99, p<0.0001). Smaller fecal sample mass resulted in a decreased diagnostic accuracy (300mg accuracy=0.77; AUC=0.69, p=0.02; 100mg accuracy=0.70, AUC=0.74, p=0.003). A loss of diagnostic accuracy was seen towards increased numbers of thaw-freeze cycles (one cycle: accuracy=0.61, AUC=0.80, p=0.0004, two cycles: accuracy=0.64, AUC=0.56, p=0.753, three cycles: accuracy=0.57, AUC=0.50, p=0.5101) and when samples were kept at room temperature for 180 minutes prior to analysis (accuracy=0.60, AUC=0.51, p=0.46). Diagnostic accuracy of VOC profiles was not significantly influenced by storage duration differences of 20 months. Conclusion Application of 500mg sample mass analyzed after one thaw-freeze cycle, showed best discriminative accuracy for differentiation of IBD and HC. VOC profiles and diagnostic accuracy were significantly affected by sampling conditions, underlining the need for implementation of standardized protocols in fecal VOC analysis

Simultaneous assessment of urinary and fecal volatile organic compound analysis in De Novo Pediatric IBD

Endoscopic evaluation is mandatory in establishing the diagnosis of pediatric inflammatory bowel disease (IBD), but unfortunately carries a high burden on patients. Volatile organic compounds (VOC) have been proposed as alternative, noninvasive diagnostic biomarkers for IBD. The current study aimed to assess and compare the potential of fecal and urinary VOC as diagnostic biomarkers for pediatric IBD in an intention-to-diagnose cohort. In this cohort study, patients aged 4–17 years, referred to the outpatient clinic of a tertiary referral center under suspicion of IBD, were eligible to participate. The diagnosis was established by endoscopic and histopathologic assessment, participants who did not meet the criteria of IBD were allocated to the control group. Participants were instructed to concurrently collect a fecal and urinary sample prior to bowel lavage. Samples were analyzed by means of gas chromatography–ion mobility spectrometry. In total, five ulcerative colitis patients, five Crohn’s disease patients, and ten age and gender matched controls were included. A significant difference was demonstrated for both fecal (p-value, area under the curve; 0.038, 0.73) and urinary (0.028, 0.78) VOC profiles between IBD and controls. Analysis of both fecal and urinary VOC behold equal potential as noninvasive biomarkers for pediatric IBD diagnosis

Detection of spontaneous preterm birth by maternal urinary volatile organic compound analysis : a prospective cohort study

Accurate prediction of preterm birth is currently challenging, resulting in unnecessary maternal hospital admittance and fetal overexposure to antenatal corticosteroids. Novel biomarkers like volatile organic compounds (VOCs) hold potential for predictive, bed-side clinical applicability. In a proof of principle study, we aimed to assess the predictive potential of urinary volatile organic compounds in the identification of pregnant women at risk for preterm birth. Urine samples of women with a high risk for preterm birth (≧24 + 0 until 36 + 6 weeks) were collected prospectively and analyzed for VOCs using gas chromatography coupled with an ion mobility spectrometer (GS-IMS). Urinary VOCs of women delivering preterm were compared with urine samples of women with suspicion of preterm birth collected at the same gestation period but delivering at term. Additionally, the results were also interpreted in combination with patient characteristics, such as physical examination at admission, microbial cultures, and placental pathology. In our cohort, we found that urinary VOCs of women admitted for imminent preterm birth were not significantly different in the overall group of women delivering preterm vs. term. However, urinary VOCs of women admitted for imminent preterm birth and delivering between 28 + 0 until 36 + 6 weeks compared to women with a high risk for preterm birth during the same gestation period and eventually delivering at term (>37 + 0 weeks) differed significantly (area under the curve: 0.70). In addition, based on the same urinary VOCs, we could identify women with a confirmed chorioamnionitis (area under the curve: 0.72) and urinary tract infection (area under the curve: 0.97). In conclusion, urinary VOCs hold potential for non-invasive, bedside prediction of preterm birth and on the spot identification of intra-uterine infection and urinary tract infections. We suggest these observations are further explored in larger populations

Profound Pathogen-Specific Alterations in Intestinal Microbiota Composition Precede Late-Onset Sepsis in Preterm Infants:A Longitudinal, Multicenter, Case-Control Study

BACKGROUND: The role of intestinal microbiota in the pathogenesis of late-onset sepsis (LOS) in preterm infants is largely unexplored but could provide opportunities for microbiota-targeted preventive and therapeutic strategies. We hypothesized that microbiota composition changes before the onset of sepsis, with causative bacteria that are isolated later in blood culture. METHODS: This multicenter case-control study included preterm infants born under 30 weeks of gestation. Fecal samples collected from the 5 days preceding LOS diagnosis were analyzed using a molecular microbiota detection technique. LOS cases were subdivided into 3 groups: gram-negative, gram-positive, and coagulase-negative Staphylococci (CoNS). RESULTS: Forty LOS cases and 40 matched controls were included. In gram-negative LOS, the causative pathogen could be identified in at least 1 of the fecal samples collected 3 days prior to LOS onset in all cases, whereas in all matched controls, this pathogen was absent (P = .015). The abundance of these pathogens increased from 3 days before clinical onset. In gram-negative and gram-positive LOS (except CoNS) combined, the causative pathogen could be identified in at least 1 fecal sample collected 3 days prior to LOS onset in 92% of the fecal samples, whereas these pathogens were present in 33% of the control samples (P = .004). Overall, LOS (expect CoNS) could be predicted 1 day prior to clinical onset with an area under the curve of 0.78. CONCLUSIONS: Profound preclinical microbial alterations underline that gut microbiota is involved in the pathogenesis of LOS and has the potential as an early noninvasive biomarker

Volatile organic compounds in breath can serve as a non-invasive diagnostic biomarker for the detection of advanced adenomas and colorectal cancer

Contains fulltext : 220031.pdf (Publisher’s version ) (Open Access)BACKGROUND: Colorectal cancer (CRC) is the third most common cancer diagnosis in the Western world. AIM: To evaluate exhaled volatile organic compounds (VOCs) as a non-invasive biomarker for the detection of CRC and precursor lesions using an electronic nose. METHODS: In this multicentre study adult colonoscopy patients, without inflammatory bowel disease or (previous) malignancy, were invited for breath analysis. Two-thirds of the breath tests were randomly assigned to develop training models which were used to predict the diagnosis of the remaining patients (external validation). In the end, all data were used to develop final-disease models to further improve the discriminatory power of the algorithms. RESULTS: Five hundred and eleven breath samples were collected. Sixty-four patients were excluded due to an inadequate breath test (n = 51), incomplete colonoscopy (n = 8) or colitis (n = 5). Classification was based on the most advanced lesion found; CRC (n = 70), advanced adenomas (AAs) (n = 117), non-advanced adenoma (n = 117), hyperplastic polyp (n = 15), normal colonoscopy (n = 125). Training models for CRC and AAs had an area under the curve (AUC) of 0.76 and 0.71 and blind validation resulted in an AUC of 0.74 and 0.61 respectively. Final models for CRC and AAs yielded an AUC of 0.84 (sensitivity 95% and specificity 64%) and 0.73 (sensitivity and specificity 79% and 59%) respectively. CONCLUSIONS: This study suggests that exhaled VOCs could potentially serve as a non-invasive biomarker for the detection of CRC and AAs. Future studies including more patients could further improve the discriminatory potential of VOC analysis for the detection of (pre-)malignant colorectal lesions. (https://clinicaltrials.gov Identifier NCT03488537)

Simultaneous Assessment of Urinary and Fecal Volatile Organic Compound Analysis in De Novo Pediatric IBD

Endoscopic evaluation is mandatory in establishing the diagnosis of pediatric inflammatory bowel disease (IBD), but unfortunately carries a high burden on patients. Volatile organic compounds (VOC) have been proposed as alternative, noninvasive diagnostic biomarkers for IBD. The current study aimed to assess and compare the potential of fecal and urinary VOC as diagnostic biomarkers for pediatric IBD in an intention-to-diagnose cohort. In this cohort study, patients aged 4–17 years, referred to the outpatient clinic of a tertiary referral center under suspicion of IBD, were eligible to participate. The diagnosis was established by endoscopic and histopathologic assessment, participants who did not meet the criteria of IBD were allocated to the control group. Participants were instructed to concurrently collect a fecal and urinary sample prior to bowel lavage. Samples were analyzed by means of gas chromatography–ion mobility spectrometry. In total, five ulcerative colitis patients, five Crohn’s disease patients, and ten age and gender matched controls were included. A significant difference was demonstrated for both fecal (p-value, area under the curve; 0.038, 0.73) and urinary (0.028, 0.78) VOC profiles between IBD and controls. Analysis of both fecal and urinary VOC behold equal potential as noninvasive biomarkers for pediatric IBD diagnosis

Risk Factors for Late-Onset Sepsis in Preterm Infants: A Multicenter Case-Control Study

Contains fulltext : 206256.pdf (publisher's version ) (Open Access